Comparing methods of determining Legionella spp. in complex water matrices.

BACKGROUND: Legionella testing conducted at environmental laboratories plays an essential role in assessing the risk of disease transmission associated with water systems. However, drawbacks of culture-based methodology used for Legionella enumeration can have great impact on the results and interpretation which together can lead to underestimation of the actual risk. Up to 20% of the samples analysed by these laboratories produced inconclusive results, making effective risk management impossible. Overgrowth of competing microbiota was reported as an important factor for culture failure. For quantitative polymerase chain reaction (qPCR), the interpretation of the results from the environmental samples still remains a challenge. Inhibitors may cause up to 10% of inconclusive results. This study compared a quantitative method based on immunomagnetic separation (IMS method) with culture and qPCR, as a new approach to routine monitoring of Legionella. RESULTS: First, pilot studies evaluated the recovery and detectability of Legionella spp using an IMS method, in the presence of microbiota and biocides. The IMS method results were not affected by microbiota while culture counts were significantly reduced (1.4 log) or negative in the same samples. Damage by biocides of viable Legionella was detected by the IMS method. Secondly, a total of 65 water samples were assayed by all three techniques (culture, qPCR and the IMS method). Of these, 27 (41.5%) were recorded as positive by at least one test. Legionella spp was detected by culture in 7 (25.9%) of the 27 samples. Eighteen (66.7%) of the 27 samples were positive by the IMS method, thirteen of them reporting counts below 10(3) colony forming units per liter (CFU l(-1)), six presented interfering microbiota and three presented PCR inhibition. Of the 65 water samples, 24 presented interfering microbiota by culture and 8 presented partial or complete inhibition of the PCR reaction. So the rate of inconclusive results of culture and PCR was 36.9 and 12.3%, respectively, without any inconclusive results reported for the IMS method. CONCLUSION: The IMS method generally improved the recovery and detectability of Legionella in environmental matrices, suggesting the possibility to use IMS method as valuable indicator of risk. Thus, this method may significantly improve our knowledge about the exposure risk to these bacteria, allowing us to implement evidence-based monitoring and disinfection strategies.

Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials.

BACKGROUND: Legionellosis is an uncommon form of pneumonia. After a clinical encounter, the necessary antibiotic treatment is available if the diagnosis is made early in the illness. Before the clinical encounter, early detection of the main pathogen involved, Legionella pneumophila, in hazardous environments is important in preventing infectious levels of this bacterium. In this study a qualitative test based on combined magnetic immunocapture and enzyme-immunoassay for the fast detection of Legionella pneumophila in water samples was compared with the standard method, in both comparative and collaborative trials. The test was based on the use of anti-Legionella pneumophila antibodies immobilized on magnetic microspheres. The final protocol included concentration by filtration, resuspension and immunomagnetic capture. The whole assay took less than 1 hour to complete. RESULTS: A comparative trial was performed against the standard culture method (ISO 11731) on both artificially and naturally contaminated water samples, for two matrices: chlorinated tap water and cooling tower water. Performance characteristics of the test used as screening with culture confirmation resulted in sensitivity, specificity, false positive, false negative, and efficiency of 96.6%, 100%, 0%, 3.4%, and 97.8%, respectively. The detection limit at the level under which the false negative rate increases to 50% (LOD50) was 93 colony forming units (CFU) in the volume examined for both tested matrices. The collaborative trial included twelve laboratories. Water samples spiked with certified reference materials were tested. In this study the coincidence level between the two methods was 95.8%. CONCLUSION: Results demonstrate the applicability of this immunosensing technique to the rapid, simple, and efficient detection of Legionella pneumophila in water samples. This test is not based on microbial growth, so it could be used as a rapid screening technique for the detection of L. pneumophila in waters, maintaining the performance of conventional culture for isolation of the pathogen and related studies.